The present invention generally concerns spectroscopic or microscopic examinations which are directed to determining the photochemical properties of observable fluorophores. With respect to relevant, related art publications, reference is made merely exemplarily to U.S. Pat. No. 5,742,437, specifically to column 9, lines 27 through 50, as well as to FIGS. 5 and 14 of the publication.
When biological specimens are examined, it is often living cells that are being observed. Fluorescent proteins are used for fluorescently tagging the cellular proteins of interest using genetic technology. Such a tagging process makes it possible to observe and analyze the protein mobility in the living cell.
What is problematic in this context is a very special phenomenon of the fluorophores, namely the occurrence of so-called dark states. This phenomenon is also called blinking and is caused, for example, by triplet transitions, isomerization, protonation, etc. The occurrence and/or frequency of such dark states, as well as their lifetimes, are dependent on numerous factors, thus, for example, on the environment of the fluorophore, but also, in particular, on the intensity of the excitation light.
The dark states, which occur regularly during observation of biological specimens, cause serious problems when their time constants are within the regions of the diffusion times, making it impossible to still distinguish between the two phenomena. In other words, it is indistinguishable whether the observed state is a dark state or the result of a cell diffusion that has occurred.